The present invention relates generally to the field of molecular biology and genetics. Specifically, the present invention relates to a method for transiently expressing a foreign gene in the cytoplasm of a plant host using a tobravirus vector.
Great interest exists in launching genome projects in human and non-human genome project. The human genome has between 2.8 million and 3.5 million base pairs, about 3 percent of which are made of genes. In June 2000, the Human Genome Project and biotech company Celera Genomics announced that a rough draft of the human genome has been completed see National Center for Biotechnology Information (NCBI) database website). This information, however, will only represent a reference sequence of the human genome. The remaining task lies in the determination of sequence functions, which are important for the study, diagnosis, and treatment of human diseases.
The Mouse genome is also being sequenced. Genbank provides about 1.2% of the 3-billion-base mouse genome (see Mouse Genome Informatics (MGI) database website) and a rough draft of the mouse genome is expected to be available by 2003 and a finished genome by 2005. In addition, the Drosophilia Genome Project has recently been completely sequenced-(see Berkeley Drosophila Genome Project database website).
Valuable and basic agricultural plants, including corn, soybeans and rice are also targets for genome projects because the information obtained thereby may prove very beneficial for increasing world food production and improving the quality and value of agricultural products. The United States Congress is considering launching a corn genome project. By helping to unravel the genetics hidden in the corn genome, the project could aid in understanding and combating common diseases of grain crops. It could also provide a big boost for efforts to engineer plants to improve grain yields and resist drought, pests, salt, and other extreme environmental conditions. Such advances are critical for a world population expected to double by 2050. Currently, there are four species which provide 60% of all human food: wheat, rice, corn, and potatoes, and the strategies for increasing the productivity of these plants is dependent on rapid discovery of the presence of a trait in these plants, and the function of unknown gene sequences in these plants.
One strategy that has been proposed to assist in such efforts is to create a database of expressed sequence tags (ESTs) that can be used to identify expressed genes. Accumulation and analysis of expressed sequence tags (ESTs) have become an important component of genome research. EST data may be used to identify gene products and thereby accelerate gene cloning. Various sequence databases have been established in an effort to store and relate the tremendous amount of sequence information being generated by the ongoing sequencing efforts. Some have suggested sequencing 500,000 ESTs for corn and 100,000 ESTs each for rice, wheat, oats, barley, and sorghum. Efforts at sequencing the genomes of plant species will undoubtedly rely upon these computer databases to share the sequence data as it is generated. Arabidopsis thaliana may be an attractive target discovery of a trait and for gene function discovery because a very large set of ESTs have already been produced in this organism, and these sequences tag more than 50% of the expected Arabidopsis genes.
Potential use of the sequence information so generated is enormous if gene function can be determined. It may become possible to engineer commercial seeds for agricultural use to convey any number of desirable traits to food and fiber crops and thereby increase agricultural production and the world food supply. Research and development of commercial seeds has so far focused primarily on traditional plant breeding, however there has been increased interest in biotechnology as it relates to plant characteristics. Knowledge of the genomes involved and the function of genes contained therein for both monocotyledonous and dicotyledonous plants is essential to realize positive effects from such technology.
The impact of genomic research in seeds is potentially far reaching. For example, gene profiling in cotton can lead to an understanding of the types of genes being expressed primarily in fiber cells. The genes or promoters derived from these genes may be important in genetic engineering of cotton fiber for increased strength or for xe2x80x9cbuilt-inxe2x80x9d fiber color. In plant breeding, gene profiling coupled to physiological trait analysis can lead to the identification of predictive markers that will be increasingly important in marker assisted breeding programs. Mining the DNA sequence of a particular crop for genes important for yield, quality, health, appearance, color, taste, etc., are applications of obvious importance for crop improvement.
Work has been conducted in the area of developing suitable vectors for expressing foreign DNA and RNA in plant and animal hosts. Ahlquist (U.S. Pat. Nos. 4,885,248 and 5,173,410) describes preliminary work done in devising transfer vectors which might be useful in transferring foreign genetic material into a plant host for the purpose of expression therein. Additional aspects of hybrid RNA viruses and RNA transformation vectors are described by Ahlquist et al. in U.S. Pat. Nos. 5,466,788, 5,602,242, 5,627,060 and 5,500,360. Donson et al., U.S. Pat. Nos. 5,316,931, 5,589,367 and 5,866,785 demonstrate for the first time plant viral vectors suitable for the systemic expression of foreign genetic material in plants. Donson et al. describe plant viral vectors having heterologous subgenomic promoters for the systemic expression of foreign genes. Carrington et al., U.S. Pat. No. 5,491,076, describe particular potyvirus vectors also useful for expressing foreign genes in plants. The expression vectors described by Carrington et al. are characterized by utilizing the unique ability of viral polyprotein proteases to cleave heterologous proteins from viral polyproteins. These include Potyviruses such as Tobacco Etch Virus. Additional suitable vectors are described in U.S. Pat. Nos. 5,811,653 and 5,977,438. Condreay, et al., (Proc. Natl. Acad. Sci. USA 96:127-132) disclose using baculoviruses to deliver and express gene efficiently in cells types of human, primate and rodent origin. Price et al., (Proc. Natl. Acad. Sci. USA 93:9465-9570 (1996)) disclose infecting insect, plant and mammalian cells with Nodaviruses.
Construction of plant RNA viruses for the introduction and expression of non-viral foreign genes in plants has also been demonstrated by Brisson et al., Methods in Enzymology 118:659 (1986), Guzman et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, pp. 172-189 (1988), Dawson et al., Virology 172:285-292 (1989), Takamatsu et al., EMBO J. 6:307-311 (1987), French et al., Science 231:1294-1297 (1986), and Takamatsu et al., FEBS Letters 269:73-76 (1990). However, these viral vectors have not been shown capable of systemic spread in the plant and expression of the non-viral foreign genes in the majority of plant cells in the whole plant. Moreover, many of these viral vectors have not proven stable for the maintenance of non-viral foreign genes. However, the viral vectors described by Donson et al., in U.S. Pat. Nos. 5,316,931, 5,589,367, and 5,866,785, Turpen in U.S. Pat. No. 5,811,653 and 5,977,438, Carrington, et al. in U.S. Pat. No. 5,491,076, have proven capable of infecting plant cells with foreign genetic material and systemically spreading in the plant and expressing the non-viral foreign genes contained therein in plant cells locally or systemically. Morsy et al., (Proc. Natl. Acad. Sci. USA, 95:7866-7871 (1998)) develop a helper-dependent adenoviral vectors having up to 37 Kb insert capacity and being easily propagated.
With the recent advent of technology for cloning, genes can be selectively turned off. One method is to create antisense RNA or DNA molecules that bind specifically with a targeted gene""s RNA message, thereby interrupting the precise molecular mechanism that expresses a gene as a protein. The antisense technology which deactivates specific genes provides a different approach from a classical genetics approach. Classical genetics usually studies the random mutations of all genes in an organism and selects the mutations responsible for specific characteristics. Antisense approach starts with a cloned gene of interest and manipulates it to elicit information about its function.
The expression of virus-derived positive sense or antisense RNA in transgenic plants provides an enhanced or reduced expression of an endogenous gene. In most cases, introduction and subsequent expression of a transgene will increase (with a positive sense RNA) or decrease (with an antisense RNA) the steady-state level of a specific gene product (Curr. Opin. Cell Biol. 7: 399-405 (1995)). There is also evidence that inhibition of endogenous genes occurs in transgenic plants containing sense RNA (Van der Krol et al., Plant Cell 2(4):291-299 (1990), Napoli et al., Plant Cell 2:279-289 (1990) and Fray et al., Plant Mol. Biol. 22:589-602 (1993)).
Post-transcriptional gene silencing (PTGS) in transgenic plants is the manifestation of a mechanism that suppresses RNA accumulation in a sequence-specific manner. There are three models to account for the mechanism of PTGS: direct transcription of an antisense RNA from the transgene, an antisense RNA produced in response to over expression of the transgene, or an antisense RNA produced in response to the production of an aberrant sense RNA product of the transgene (Baulcombe, Plant Mol. Biol 32:79-88 (1996)). The posttranscriptional gene silencing mechanism is typified by the highly specific degradation of either the transgene mRNA or the target RNA, by RNA having either the same or complementary nucleotide sequences. In cases that the silencing transgene is the same sense as the target endogenous gene or viral genomic RNA, it has been suggested that a plant-encoded RNA-dependent RNA polymerase makes a complementary strand from the transgene mRNA and that the small cRNAs potentiate the degradation of the target RNA. Antisense RNA and the hypothetical cRNAs have been proposed to act by hybridizing with the target RNA to either make the hybrid a substrate for double-stranded (ds) RNases or arrest the translation of the target RNA (Baulcombe, Plant Mol. Biol. 32: 79-88 (1996)). It is also proposed that this downregulation or xe2x80x9cco-suppressionxe2x80x9d by the sense RNA might be due to the production of antisense RNA by readthrough transcription from distal promoters located on the opposite strand of the chromosomal DNA (Grierson et al., Trends Biotechnol. 9:122-123 (1993)).
Kumagai, et al. (Proc. Natl., Acad. Sci. USA 92:1679 (1995)) report that gene expression in transfected Nicotiana benthamiana was cytoplasmic inhibited by viral delivery of a RNA of a known sequence derived from cDNA encoding tomato phytoene desaturase in a positive sense or an antisense orientation. The plant host, Nicotiana benthamiana, and the donor plant, tomato (Lycopersicon esculentum), belong to the same family. There is also evidence that inhibition of endogenous genes occurs in transgenic plants containing sense RNA (Van der Krol et al., Plant Cell 2(4):291-299 (1990), Napoli et al., Plant Cell 2:279-289 (1990) and Fray et al., Plant Mol. Biol. 22:589-602 (1993)).
U.S. Pat. No. 5,922,602 (Kumagai, et al.) discloses a silencing vector comprising dual subgenomic promoters. Kumagai, et al. teach a genetic vector comprising: (a) a first viral subgenomic promoter operably joined to a first nucleic acid sequence that codes for a plant viral coat protein wherein the transcription of the first nucleic acid sequence is regulated by the first plant viral subgenomic promoter; (b) a second plant viral subgenomic promoter operably joined to a second nucleic acid sequence which codes for an anti-sense RNA or a co-suppressor RNA specific for a gene of interest in a plant wherein transcription of the second nucleic acid sequence is regulated by the second plant viral subgenomic promoter; and (c) an origin of replication that initiates replication of the genetic vector in the cytoplasm of a plant cell.
WO 99/36516 (BIOSOURCE TECHNOLOGIES, INC.) discloses a method of determining the function of nucleic acid sequences, changing the phenotypic or biochemical characteristics, and silencing endogenous genes by transfecting a plant host with a recombinant viral nucleic acid comprising a foreign nucleic acid sequence. The recombinant viral nucleic acid is derived from a monopartite plus sense single-stranded RNA virus.
MacFarlane and Popovich (Virology 267:29-35 (2000)) constructed viral vectors from infectious cDNA clones of each of the three tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP). The TRV-GFP construct was prepared by removal of 3xe2x80x2 part of the 2b gene and the entire 2c gene. The PEBV-GFP construct was prepared by removal of 2b and 2c genes. The PepRSV-GFP was prepared by removal of 3xe2x80x2 part of the 2b gene and the entire 2c gene. The modified RNA2 constructs that MacFarlane and Popovich teach do not have the entire 2b gene.
The present invention provides a method for either silencing an endogenous gene of a plant host or expressing a foreign gene in a plant host using a monopartite or a bipartite plant viral vector derived from a tobravirus.
This invention is directed to a bipartite RNA viral vector comprising: (a) modified tobravirus RNA-1 comprising an inserted foreign RNA sequence that codes for all or part of a protein; the inserted sequence is operably linked to the 3xe2x80x2 end of the stop codon of the RNA sequence that codes for a 16 k Da cysteine-rich protein of RNA-1; and (b) tobravirus RNA-2. The tobravirus RNA-2 may comprise a promoter-gene construct inserted in place of the 2C gene, wherein the promoter-gene construct comprises a second foreign RNA sequence.
The invention is also directed to a monopartite RNA viral vector comprising: modified tobravirus RNA-1 comprising an inserted foreign RNA sequence; the inserted sequence is operably linked to the 3xe2x80x2 end of the stop codon of the RNA sequence which codes for a 16 k Da cysteine-rich protein of RNA-1. The foreign RNA is either in a positive sense or an anti-sense orientation, and is either a complete open reading frame or a partial open reading frame.
The invention is directed to a method of simultaneously silencing one or more endogenous host genes and a method of simultaneously silencing a plant host gene and expressing a foreign gene. Such method comprises infecting a plant host with a bipartite vector comprising tobravirus RNA-1 and RNA-2, wherein the RNA-1 comprises a first foreign RNA sequence that codes for all or part of a first protein, and the RNA-2 comprises a second foreign RNA sequence that codes for all or part of a second protein.
The invention is further directed to a method of compiling a plant functional gene profile, a method of changing the phenotype or biochemistry of a plant host, and a method of determining the presence of a trait in a plant host. Such methods comprise the steps of preparing a library of DNA or RNA sequences from a donor plant, and constructing recombinant viral nucleic acids comprising an unidentified nucleic acid insert obtained from said library, wherein said recombinant viral nucleic acids are obtained from a tobravirus.